Fusion of Pinoresinol-lariciresinol reductase with Secoisolariciresinol dehydrogenase for converting Pinoresinol into Matairesinol

• Main Authors: Zhi-Yu Wei, 魏至宇
• Other Authors: 李昆達
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/25169706539952605841

碩士 === 國立臺灣大學 === 生化科技學系 === 99 === Lignans, a class of dimeric phenylpropanoid derivatives, existing in whole-grain, sesame and flax seeds show anticancer activity and can react as phytoestrogen. Secoisolariciresinol and matairesinol, members of lignans, can be converted to enterolactone and enterodiol in mammalian proximal colon. Containing high concentration of enterolactone in serum and urine has positive effect for the treatment of breast cancer, osteoporosis and colon cancer. To establish a continuous bioconversion system for matairesinol formation from pinoresinol, pinoresinol reductase (plr) and secoisolariciresinol dehydrogenase (sdh) which were cloned from Podophyllum peltatum Hance were linked by asymmetric overlap extension by polymerase chain reaction (AOE-PCR), to form a bifunctional fusion gene (plr-sdh) and was expressed in Escherichia coli. The optimized conditions for recombinant fusion protein induction in E.coli were 0.01 mM IPTG, 12 h, 25oC determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. At pH 8.0, 22OC, 1 h reaction in vitro the results showed that the bioconversion efficiency of fusion protein PLR-SDH was higher than the mixture of individual rPLR and rSDH. The bioconversion rate of PLR-SDH was 49.82%, and 3.41% /